This invention relates to novel semi-synthetic antifungal compounds which are prepared by the acylation of the cyclic peptide nucleus produced by the enzymatic deacylation of antibiotic A30912 factor D.
Antibiotic A-30912 factor D is an antifungal cyclic peptide having the formula: ##STR10## wherein R is the linoleoyl group ##STR11## Throughout this application, the cyclic peptide formulas, such as Formula I, assume that the amino acids represented are in the L-configuration. The factor is isolated from the A30912 complex which contains other factors arbitrarily designated factors A, B, C, E, F, and G. The A-30912 complex and the individual factors A through G are disclosed by M. Hoehn and K. Michel in U.S. Pat. No. 4,024,245. Antibiotic A-30912 factor A is identical to antibiotic A-22802 which is described by C. Higgins and K. Michel in U.S. Pat. No. 4,024,246. Factor D has also been found to be identical to antibiotic echinocandin D [see R. Traber et al., Helv. Chim. Acta, 62, 1252 (1979)] and to antibiotic SL 7810/F-III [see Belgium Patent No. 834, 289, Derwent Abstract 30159X].
Antibiotic A-30912 factor D is prepared by fermentation using one of several different organisms, namely: (a) Aspergillus rugulosus NRRL 8113 (see U.S. Pat. No. 4,024,245); (b) Aspergillus nidulans var. echinulatus A-32204, NRRL 3860, as described in Swiss Patent No. 568,386; (c) Aspergillus rugulosus NRRL 8039 (see Belgian Patent No. 834,289); or (d) Aspergillus nidulans var. roseus NRRL 11440 (see co-pending application of L. Boeck and R. Kastner, METHOD OF PRODUCING THE A-30912 ANTIBIOTICS, Ser. No. 126,078, filed Mar. 3, 1980, which is a continuation-in-part of application Ser. No. 46,744, filed June 8, 1979, (now abandoned), the entire disclosure of which is incorporated herein by reference).
A subculture of A. nidulans var. roseus has been deposited and made a part of the permanent culture collection of the Northern Regional Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, Peoria, Illinois 61604, from which it is available to the public under the number NRRL 11440.
When a strain of A. nidulans var. roseus NRRL 11440 is used to produce A-30912 factor D, a complex of factors is obtained which for convenience is called the A-42355 antibiotic complex. A-30912 factor A is the major factor of the A-42355 antibiotic complex, while factors B, D and H are minor factors. Examples 5, 6, and 7 herein, illustrate the preparation of the A-42355 complex and the isolation and purification of A-30912 factor D therefrom. A-30912 factor H is further described in a co-pending application of Karl H. Michel entitled ANTIBIOTIC A-30912 FACTOR H, Ser. No. 117,739, filed Feb. 1, 1980, which is a continuation-in-part of application Ser. No. 46,875, filed June 8, 1979 (now abandoned).
In the antibiotic A-30912 factor D molecule (Formula I), the linoleoyl side chain (R) is attached at the cyclic peptide nucleus at the .alpha.-amino group of the ornithine residue. Surprisingly, it has been found that the linoleoyl side chain can be cleaved from the nucleus by an enzyme without affecting the chemical integrity of the nucleus. The enzyme employed to effect the deacylation reaction is produced by a microorganism of the family Actinoplanaceae, preferably the microorganism Actinoplanes utahensis NRRL 12052, or a variant thereof. To accomplish deacylation, antibiotic A30912 factor D is added to a culture of the microorganism and the culture is allowed to incubate with the substrate until the deacylation is substantially complete. The cyclic nucleus thereby obtained is separated from the fermentation broth by methods known in the art. Unlike antibiotic A-30912 factor D, the cyclic nucleus (lacking the linoleoyl) side chain) is substantially devoid of antifungal activity.
The cyclic nucleus afforded by the aforedescribed enzymatic deacylation of antibiotic A-30912 factor D, is depicted in Formula II. ##STR12##
A-30912D nucleus has an empirical formula of C.sub.34 H.sub.51 N.sub.7 O.sub.12 and a molecular weight of 749.83.
Removal of the side chain group affords a free primary .alpha.-amino group in the ornithine residue of the cyclic peptide. For convenience, the compound having the structure given in FORMULA II will be referred to herein as "A-30912D nucleus." As will be apparent to those skilled in the art, A-30912D nucleus can be obtained either in the form of the free amine or of the acid addition salt. Although any suitable acid addition salt may be employed, those which are non-toxic and pharmaceutically acceptable are preferred.
The method of preparing A-30912D nucleus from antibiotic A-30912 factor D by means of fermentation using Actinoplanes utahensis NRRL 12052 is described in the co-pending application of Bernard J. Abbott and David S. Fukuda, entitled "A-30912D NUCLEUS", Docket No. X-5188, Ser. No. 103,268, which was filed Dec. 13, 1979. A continuation-in-part application of this application, with the corresponding Docket No. X-5188A, is being filed herewith this even date, the full disclosure of which is incorporated herein by reference. Example 3 herein, illustrates the preparation of A-30912D nucleus by fermentation using antibiotic A-30912 factor D as the substrate and Actinoplanes utahensis NRRL 12052 as the microorganism.
The enzyme produced by Actinoplanes utahensis NRRL 12052 may be the same enzyme which has been used to deacylate penicillins (see Walter J. Kleinschmidt, Walter E. Wright, Frederick W. Kavanagh, and William M. Stark, U.S. Pat. No. 3,150,059, issued Sept. 22, 1964).
Cultures of representative species of Actinoplanaceae are available to the public from the Northern Regional Research Laboratory under the following accession numbers:
______________________________________ Actinoplanes utahensis NRRL 12052 Actinoplanes missouriensis NRRL 12053 Actinoplanes sp. NRRL 8122 Actinoplanes sp. NRRL 12065 Streptosporangium roseum var. hollandensis NRRL 12064 ______________________________________
The effectiveness of any given strain of microorganism within the family Actinoplanaceae for carrying out the deacylation of this invention is determined by the following procedure. A suitable growth medium is inoculated with the microorganism. The culture is incubated at about 28.degree. C. for two or three days on a rotary shaker. One of the substrate antibiotics is then added to the culture. The pH of the fermentation medium is maintained at about pH 6.5. The culture is monitored for activity using a Candida albicans assay. Loss of antibiotic activity is an indication that the microorganism produces the requisite enzyme for deacylation. This must be verified, however, using one of the following methods: (1) analysis by HPLC for presence of the intact nucleus; or (2) reacylation with an appropriate side chain (e.g. linoleoyl, stearoyl, or palmitoyl) to restore activity.
It is known that another antibiotic substance possesses the same nucleus as that of antibiotic A-30912 factor D. This substance, which differs from antibiotic A-30912 factor D in that a different acyl group is present in place of the linoleoyl group (R) in Formula I, is tetrahydro-A-30912 factor D (tetrahydro-SL 7810/F-III; tetrahydro echinocandin D) described by R. Traber et al., Helv. Chim. Acta, 62 1252 (1979). Tetrahydro-A-30912 factor B is depicted in Formula I when R is stearoyl. Tetrahydro-A-30912 factor D can be prepared from antibiotic A-30912 factor D by catalytic hydrogenation using PtO.sub.2 in ethanol under positive pressure. Tetrahydro-A-30912 factor D can be employed as a substrate in place of antibiotic A-30912 factor D for the enzymatic deacylation using the procedure herein described.